Methylation of cytosines within CpG dinucleotides is critical for normal development in mammals. In cancer, proper regulation of DNA methylation goes awry often leading to silencing of tumor suppressors or activation of growth-stimulating genes. These events can strongly contribute to tumorigenesis. Many loci have been identified that acquire methylation or whose expression is methylation-sensitive in the normal and cancer genome, but virtually nothing is known about how methylation is regulated. Imprinted loci are useful for identifying cis-acting DNA sequences that regulate local methylation since these loci undergo predictable patterns of allele-specific methylation in normal tissue. RASGRF1 is a GTP exchange factor that activates RAS and has transforming activity. In mice, the Rasgrf1 locus is imprinted: There is paternal allele-specific methylation within a differentially methylated domain (DMD) 30 kbp 5' of the promoter and the locus is paternally expressed. We have shown that a repeated sequence element found immediately 3' of the DMD regulates establishment of methylation of the DMD in the male germ line. The DMD behaves like a methylation-sensitive enhancer-blocking element and together with the repeat sequence, represents a binary switch that regulates allele-specific expression of the locus. The central goal of this proposal is to elaborate the mechanisms by which the repeat element regulates DNA methylation. If we understand how DNA methylation is normally regulated, this may help us understand how inappropriate methylation occurs in cancer. Furthermore, these studies may identify therapeutic targets for treating diseases characterized by aberrant DNA methylation.